A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan

The rapid identification of Influenza A virus and its variants, which cause severe respiratory diseases, is imperative to providing timely treatment and improving patient outcomes. Conventionally, two separate assays (total test duration of up to 6 h) are required to initially differentiate Influenza A and B viruses and subsequently distinguish the pdm H1N1 and H3N2 serotypes of Influenza A virus. In this study, we developed a multiplex real-time RT-PCR method for simultaneously detecting Influenza A and B viruses and subtyping Influenza A virus, with a substantially reduced test duration. Clinical specimens from hospitalized patients and outpatients with influenza-like symptoms in Eastern Taiwan were collected between 2011 and 2015, transported to Hualien Tzu Chi Hospital, and analyzed. Conventional RT-PCR was used to subtype the isolated Influenza A viruses. Thereafter, for rapid identification, the multiplex real-time RT-PCR method was developed and applied to identify the conserved regions that aligned with the available primers and probes. Accordingly, a multiplex RT-PCR assay with three groups of primers and probes (MAF and MAR primers and MA probe; InfAF and InfAR primers and InfA probe; and MBF and MBR primers and MB probe) was established to distinguish these viruses in the same reaction. Thus, with this multiplex RT-PCR assay, Influenza B, Influenza A pdm H1N1, and Influenza A H3N2 viruses were accurately detected and differentiated within only 2.5 h. This multiplex RT-PCR assay showed similar analytical sensitivity to the conventional singleplex assay. Further, the phylogenetic analyses of our samples revealed that the characteristics of these viruses were different from those reported previously using samples collected during 2012–2013. In conclusion, we developed a multiplex real-time RT-PCR method for highly efficient and accurate detection and differentiation of Influenza A and B viruses and subtyping Influenza A virus with a substantially reduced test duration for diagnosis.

3 Response: We have removed the funding-related text from the manuscript. Also, please update our Funding Statement in the online submission, which should read as: "This work was supported by Grants from the Centers for Disease Control, R.O.C (Taiwan).
(HZ099103, HZ100102, HZ101060, CH102055, CW103035)." 4 Responses to Reviewer 1: Comment 1: The manuscript has to be revised for English language, there is several words used which is giving another meaning and impression like: (c) Line 319: Influenza virus genome segment "Develops" , "evolve" would be the correct word.
Response to (c): Thank you. We have replaced the word by "evolve" (line 329, unmarked version).
(d) the mistakes with the English Language is repeated in several sections of the MS so i would recommend professional English editing before publication.

Response to (d):
In response to the recommendation from the reviewer, we have asked help to improve the language of our revised manuscript by a professional English editor (KGSupport). The certificate of this English editing service is provided at the end of this document.

Response:
We thank the reviewer for this suggestion. The misleading term has now been replaced by "viral cDNA sequencing" (lines 193 and 194, unmarked version).

Comment 4:
No statistics made, Statistical analysis has to be made to address the significance and compare the sensitivity of the 2 sets of primers and probes for better conclusion.

Response:
We thank the reviewer for this excellent suggestion. In response to this comment, we have additionally provided data of analytical sensitivity of the conventional singleplex and our multiplex assays analyzed by standard curves of CT values. For comparisons of CT values and Pearson correlation coefficients between the 6 conventional singleplex and our multiplex assays, we have used independent sample ttests and no significances were detected. Thus, we have made the following revisions: (1) This additional set of data is presented as Table 5 (page 29, unmarked version).
(2) The results of these analyses have been reported in the Result section (lines 248-254 , unmarked version). These statements read as:" Table 5 shows the data of the analytical sensitivity of the multiplex assays developed in this study and the traditional singleplex assays. The CT values for each standard concentration obtained from the use of MAF/R, MBF/R, and InfAF/R in the multiplex assays did not significantly differ from those in the singleplex assays. Further analyses of standard curves of CT values revealed that the correlation coefficients, slopes, and amplification efficiencies of these two types of assays had no significant differences." (3) The results of these analyses have been reported in the abstract (lines 52-53, unmarked version) and the statement reads as: "The analytical sensitivity of this multiplex RT-PCR assay was as good as that of the conventional singleplex assay." (4) A paragraph of statistical analysis has been added to the revised manuscript (lines

224-230, unmarked version).
We sincerely hope that the reviewer could approve our explanations. 7 Responses to Reviewer 2: Comment 1: As mentioned in this paper the aim of this project was avoid the false negatives caused using the WHO-recommended primers and probes for detecting influenza A and B viruses, and to shorten total time needed to differentiate influenza A and B. Authors must be clarified this time under 2.5 hours, is include the time of viral RNA extraction? If is include, how many samples?

Response:
We thank the reviewer for reminding us this important issue. The time of viral RNA extraction was not included in the test duration of 2.5 hours, which was indicated only for the multiplex RT-PCR assay. In response to the reviewer's comment, we have added a statement in the revised manuscript to clarify this issue (lines 370-371, unmarked version). The statement reads as; "The total test duration did not include the time of viral RNA extraction." Comment 2: Considering that there are currently rapid one-step commercial kits and some papers, for detecting several respiratory viruses at the same time, authors must be comprised their methods with these commercial kits in the discussion section of the article. such as: A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses Response: We thank the reviewer for providing the valuable reference, which has been added to our reference list (ref. 25). In response to this comment, we have added a paragraph (lines 378-385, unmarked version) to discuss the methods of that work (ref. 25

TO WHOM IT MAY CONCERN:
This memo certifies that Li-Kuang Chen, the author(s) of the below mentioned document had contracted our English language document review service for one (1) file, the particulars of which are listed below. The English review was conducted using a two-stage process, in which two editors reviewed the file, both of whom are native English speakers.
We therefore attest to the quality of the reviewed document.

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September 24, 2022 A highly efficient and accurate method of detecting and subtyping Influenza A pdm H1N1 and H3N2 viruses with newly emerging mutations in the matrix gene in Eastern Taiwan